MolPhase: An Advanced Phase Separation Predictor and an Investigation of Phytobacterial Effector in Plant

Abstract

We introduce MolPhase (http://molphase.sbs.ntu.edu.sg/), an advanced protein phase separation (PS) prediction algorithm that improves accuracy and reliability by utilizing diverse physicochemical features and extensive experimental datasets. MolPhase applies a user-friendly interface to compare distinct biophysical features side-by-side along protein sequences. By additional comparison with structural predictions, MolPhase enables efficient predictions of new phase-separating proteins and guides hypothesis generation and experimental design. Key contributing factors underlying MolPhase include pi-pi interaction, disorder, and prion-like domain. As an example, MolPhase finds that phytobacterial type III effectors (T3Es) are highly prone to homotypic PS, which was experimentally validated in vitro biochemically and in vivo in plants, mimicking their injection and accumulation in the host during microbial infection. In addition, the phase-separation of T3Es were evolved both in vivo and in vitro, suggesting their determinative scaffolding function, though there is a difference in material properties, implying a difference in homotypic and heterotypic macromolecular condensation. Robust integration of MolPhase's effective prediction and experimental validation exhibit the potential to evaluate and explore how biomolecule PS functions in biological systems.

Phase-separating MoSpa2 Complex Organizes Actin Nucleation Center forM.oryzaePlant Infection

“…To investigate how the MoSpa2 domain regulates actin polymerization and stabilization, we conducted a series of in vitro biochemical experiments using various recombinant MoSpa2 variants. Employing the E. coli expression system, we generated four distinct MoSpa2 variants and the full-length protein, guided by the Alphafold2 prediction and residue signature analysis (Liang et al , 2023) (Fig 3A and B; Appendix Fig S4A and B). The developed protein variants are MoSpa2(1-747), MoSpa2(1-338), MoSpa2(339-747), and MoSpa2(748-951) (Appendix Fig S4C).…”

“…The C-terminal region of MoBni1 (1002-1740aa, MoBni1FH1C), encompassing the biocatalytic domains of polyP-containing FH1, dimeric FH2, and C-terminal IDR, was characterized using Alphafold and distinct physiochemical motif analysis through the machine-learning phase separation engine MolPhase (Graziano et al , 2011a; Liang et al ., 2023; Ma et al , 2022a; Miao et al ., 2013; Moseley & Goode, 2005b; Moseley & Goode, 2006; Sun et al , 2018; Xie et al , 2020; Xie et al ., 2019; Xie et al ., 2022) (Fig 3D; Appendix Fig S6A and B). Then, we generatd biochemically active MoBni1 variant MoBni1FH1C (Appendix Fig S6C) and examined its functional interplay with MoSpa2 variants.…”

“…To identify the MoSpa2 sequence characteristics and MoBni1sequence features, analysis of MoSpa2 truncates sites, full length sequence of MoSpa2 and MoBni1 sequence were submitted to MolPhase, which is a novel engine including all below individual traits(Liang et al ., 2023). ANCHOR (http://anchor.enzim.hu/) for identifying IDR domains.…”

“…The C-terminal region of MoBni1 (1002-1740aa, MoBni1FH1C), encompassing the biocatalytic domains of polyP-containing FH1, dimeric FH2, and C-terminal IDR, was characterized using Alphafold and distinct physiochemical motif analysis through the machine-learning phase separation engine MolPhase (Graziano et al, 2011a;Liang et al, 2023;Ma et al, 2022a;Miao et al, 2013;Moseley & Goode, 2005b;Moseley & Goode, 2006;Sun et al, 2018;Xie et al, 2020;Xie et al, 2019;Xie et al, 2022) We then investigated how the phase separation-mediated multivalent interactions of MoBni1 influenced its actin nucleation activities in the presence of different MoSpa2 truncating variants. Using the TIRF actin polymerization assay with 2.5 nM MoBni1-FH1C, we observed that MoBni1FH1C efficiently nucleated actin assembly and generated short Factin seeds on its own.…”

“…To identify the MoSpa2 sequence characteristics and MoBni1sequence features, analysis of MoSpa2 truncates sites, full length sequence of MoSpa2 and MoBni1 sequence were submitted to MolPhase, which is a novel engine including all below individual traits (Liang et al, 2023) For MoSpa2 variants phase diagram, a serial dilution of protein concentration and ionic strength was performed. Volume of 10 L serial dilution protein in different dose NaCl solution were dropped onto coverslip for 5 minutes before images.…”